Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration

The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.     

The LG3 module of laminin-5 harbors a binding site for integrin alpha3beta1 that promotes cell adhesion, spreading, and migration

Laminins are a family of extracellular matrix glycoproteins involved in cell adhesion and migration. A major obstacle to understanding their structure-function relationships is the lack of small laminin domains capable of replicating integrin-binding, cell-adhesive, and migratory functions of the intact molecule. Here, we show that the recombinant LG3 (rLG3) module (26 kDa) of laminin-5 (Ln-5) alpha(3) chain replicated key Ln-5 activities. rLG3 but not rLG1 or rLG2 supported cell adhesion and migration of at least two distinct cell lines, in an integrin alpha(3)beta(1)-dependent manner. Cell adhesion to rLG3 was regulated by divalent cations and accompanied by cell spreading and tyrosine phosphorylation of FAK focal adhesion kinase. The integrin binding activity of rLG3 was confirmed by rLG3 affinity chromatography of detergent cell lysates, which resulted in specific purification of integrin alpha(3)beta(1). To our knowledge, this is the first report directly demonstrating that a recombinant laminin LG module is an active domain capable of supporting integrin-dependent cell adhesion and migration.     

Threonine 788 in integrin subunit beta1 regulates integrin activation

In the present study, the functional role of suggested phosphorylation of the conserved threonines in the cytoplasmic domain of integrin subunit beta1 was investigated. Mutants mimicking phosphorylated and unphosphorylated forms of beta1 were expressed in beta1 deficient GD25 cells. T788 in beta1 was identified as a site with major influence on integrin function. The mutation to A788 strongly reduced beta1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate integrin activation, but the talin head domain bound equally well to the wild-type beta1 and the mutants indicating that the T788A mutation caused defect integrin activation by another mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting cell adhesion. GD25 cells expressing beta1T788D accumulated increased number of focal contacts and migrated slowly compared to GD25 beta1 wild-type. An analogous phenotype is seen when focal adhesion kinase activation is abrogated. However, neither the beta1T788D nor the beta1T788A mutation failed to induce tyrosine phosphorylation of focal adhesion kinase. The results suggest that phosphorylation of T788 in integrin beta1 promotes inside-out receptor activation, as well as focal contact accumulation.     

The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 alpha3 chain is crucial for integrin alpha3beta1 binding and cell adhesion

Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin alpha3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin alpha3beta1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin alpha3beta1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin alpha3beta1-dependent cell adhesion and spreading.     

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.     

Phosphorylation of beta3 integrin controls ligand binding strength.

The cytoplasmic domain of beta(3) integrin contains tyrosines at positions 747 and 759 in domains that have been implicated in regulation of alpha(v)beta(3) function and that serve as potential substrates for Src family kinases. The phosphorylation level of beta(3) integrin was modulated using a temperature-sensitive v-Src kinase. Increased beta(3) phosphorylation abolished alpha(v)beta(3)- but not alpha(5)beta(1)-mediated adhesion to fibronectin. alpha(v)beta(3)-Mediated cell adhesion was restored by the expression of beta(3) containing Y747F or Y759F mutations but not by wild type beta(3) integrin. Thus, phosphorylation of the cytoplasmic domain of beta(3) is a negative regulator of alpha(v)beta(3)-fibronectin binding strength.     

The cytoplasmic domain of the integrin alpha9 subunit requires the adaptor protein paxillin to inhibit cell spreading but promotes cell migration in a paxillin-independent manner

The integrin alpha9 subunit forms a single heterodimer, alpha9beta1. The alpha9 subunit is most closely related to the alpha4 subunit, and like alpha4 integrins, alpha9beta1 plays an important role in leukocyte migration. The alpha4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the alpha9 cytoplasmic domain has similar effects, a series of chimeric and deleted alpha9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an alpha9beta1-specific ligand. Like alpha4, the alpha9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the alpha9 cytoplasmic domain and to a similar level as alpha4. In paxillin(-/-) cells, alpha9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the alpha9-paxillin interaction prevented alpha9 from inhibiting cell spreading but had no effect on alpha9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin alpha subunits enhance migration and inhibit cell spreading are distinct and that the alpha9 and alpha4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.     

ADAM15 suppresses cell motility by driving integrin alpha5beta1 cell surface expression via Erk inactivation

Human ADAM15 is unique among the A disintegrin and metalloprotease domain (ADAM) family because of the integrin binding motif Arg-Gly-Asp (RGD) within its disintegrin domain. Integrin alpha5beta1 has been reported to bind to ADAM15 in an RGD-dependent manner, but the biological significance of the interaction between ADAM15 and alpha5beta1 is unknown. To characterize the effects of ADAM15 on alpha5beta1-mediated cell adhesion and migration and elucidate the potential mechanism, CHO cells which express endogenous integrin alpha5beta1 were transfected with human ADAM15 cDNA. ADAM15 overexpression led to enhanced cell adhesion and decreased migration on fibronectin, which were suppressed by down-regulation of integrin alpha5. Overexpression of ADAM15 not only increased the cell surface expression of integrin alpha5 but also resulted in a more clustered staining of alpha5 on cell surface, while the beta1 subunit remained unchanged. Unexpectedly, results from immunoprecipitation and immunofluorescence indicated that ADAM15 and alpha5beta1 integrin did not interact directly in CHO cells. We found that ADAM15 expression decreased the phosphorylation of Erk1/2. Consistently, down-regulation of Erk1/2 phosphorylation by MEK inhibitor PD98059 or siRNA against Erk1/2 enhanced the expression of alpha5 on cell surface. By using a B16F10 pulmonary metastasis model, we revealed that overexpression of ADAM15 significantly reduced the number of metastatic nodules on the lung. Taken together, this study reveals for the first time that ADAM15 could drive alpha5 integrin expression on cell surface via down-regulation of phosphorylated Erk1/2. This presents a novel mechanism by which ADAM15 regulates cell-matrix adhesion and migration.     

Dual functionality of the anti-beta1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.     

A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function.

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin alphavbeta3 inhibits both phagocytosis and migration mediated by alpha5beta1 on the same cell, and the beta3 cytoplasmic tail is necessary and sufficient for this regulation of alpha5beta1. Ligation of alpha5beta1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for alpha5beta1-mediated phagocytosis and migration. Simultaneous ligation of alphavbeta3 or expression of a chimeric molecule with a free beta3 cytoplasmic tail prevents alpha5beta1-mediated activation of CamKII. Expression of a constitutively active CamKII restores alpha5beta1 functions blocked by alphavbeta3-initiated integrin crosstalk. Thus, alphavbeta3 inhibition of alpha5beta1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the beta3 cytoplasmic tail demonstrates a requirement for Ser752 in beta3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not beta3 tyrosine phosphorylation in initiation of the alphavbeta3 signal for integrin crosstalk.     

Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2

Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).     

Binding of Paxillin to the alpha 9 Integrin Cytoplasmic Domain Inhibits Cell Spreading

alpha(9)beta(1) integrin is a member of the beta(1) integrin family, plays an important role in extravasation of neutrophils at sites of acute inflammation, and is required for the normal development of the lymphatic system. The alpha(9) and alpha(4) integrin subunits are most closely related and form a subfamily of integrin alpha subunits. Previously, we have reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule. This interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion, including stimulation of cell migration and inhibition of cell spreading and focal adhesion formation. In the current studies, we have examined the interaction between the alpha(9) cytoplasmic domain and paxillin. Here we report that the alpha(9) cytoplasmic domain binds paxillin directly and tightly and that the alpha(9)-paxillin association inhibits cell spreading. We have identified amino acid residues in the alpha(9) cytoplasmic domain, Trp(999) and Trp(1001), that are critical for paxillin binding, and alanine substitution of either Trp(999) or Trp(1001) blocks paxillin binding. Furthermore, these mutations also reverse the effect of the alpha(9) cytoplasmic domain on cell spreading. Thus, the alpha(9) and alpha(4) integrin subunits form a paxillin-binding subfamily of integrin alpha subunits, and direct binding of paxillin to the alpha(9) cytoplasmic domain mediates some of the biological activities of the alpha(9)beta(1) integrin.     

P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.     

CYR61 stimulates human skin fibroblast migration through Integrin alpha vbeta 5 and enhances mitogenesis through integrin alpha vbeta 3, independent of its carboxyl-terminal domain

CYR61, an angiogenic factor and a member of the CCN protein family, is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, promotes cell migration, and enhances growth factor-stimulated cell proliferation. CYR61 induces angiogenesis and promotes tumor growth in vivo and is expressed in dermal fibroblasts during cutaneous wound healing. It has been demonstrated recently that adhesion of primary skin fibroblasts to CYR61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, resulting in adhesive signaling and up-regulation of matrix metalloproteinases 1 and 3. CYR61 is composed of four discrete structural domains that bear sequence similarities to the insulin-like growth factor-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a carboxyl-terminal (CT) domain that resembles cysteine knots found in some growth factors. In this study, we show that a CYR61 mutant (CYR61DeltaCT) that has the CT domain deleted is unable to support adhesion of primary human skin fibroblasts but is still able to stimulate chemotaxis and enhance basic fibroblast growth factor-induced mitogenesis similar to wild type. In addition, fibroblast migration to CYR61 is mediated through integrin alpha(v)beta(5) but not integrins alpha(6)beta(1) or alpha(v)beta(3). Furthermore, we show that CYR61 binds directly to purified integrin alpha(v)beta(5) in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin alpha(v)beta(3), a known receptor for CYR61 that mediates CYR61-dependent cell adhesion and chemotaxis in vascular endothelial cells. Thus, CYR61 promotes primary human fibroblast adhesion, migration, and mitogenesis through integrins alpha(6)beta(1), alpha(v)beta(5), and alpha(v)beta(3), respectively. Together, these findings establish CYR61 as a novel ligand for integrin alpha(v)beta(5) and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner.     

Transmembrane domain helix packing stabilizes integrin alphaIIbbeta3 in the low affinity state

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.     

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.     

Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by alpha 3beta 1 integrin in oral keratinocytes

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.     

R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells.

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the alpha2, but not the alpha5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin beta cytoplasmic domains was observed for the alpha2 cytoplasmic domain; alpha2beta1-mediated migration was inhibited by the expression of excess alpha2 but not alpha5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin alpha subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin alpha cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.     

Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction.     

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.